Masp-2, a complement fixing enzyme, and uses for it

ABSTRACT

The invention relates to the discovery and characterization of mannan binding lectin-associated serine protease-2 (MASP-2), a new serine protease that acts in the MBLectin complement fixation pathway.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation (and claims the benefit of priorityunder 35 U.S.C. §120) of U.S. patent application Ser. No. 11/518,398,filed Sep. 8, 2006, now pending, which is a continuation of U.S. patentapplication Ser. No. 11/314,817, filed Dec. 21, 2005, now pending, whichis a continuation of U.S. patent application Ser. No. 09/874,238, filedJun. 4, 2001, now issued as U.S. Pat. No. 7,083,786, which is acontinuation of U.S. patent application Ser. No. 09/054,218, filed Apr.2, 1998, now abandoned, which claims the benefit under 35 U.S.C. §119(e)of U.S. Provisional Application No. 60/042,678, filed Apr. 3, 1997, nowexpired, all hereby incorporated by reference.

FIELD OF THE INVENTION

The invention is in the general field of innate pathways for complementfixation involving mannan-binding lectin (MBL), also termed mannanbinding protein.

BACKGROUND OF THE INVENTION

The complement system comprises a complex array of enzymes andnon-enzymatic proteins of importance to the function of the innate aswell as the adaptive immune defense¹. Until recently two modes ofactivation were known, the classical pathway initiated byantibody-antigen complexes and the alternative pathway initiated bycertain structures on microbial surfaces. A third, novelantibody-independent pathway of complement activation has beendescribed². This pathway is initiated when mannan-binding lectin (MBL,first described as mannan-binding protein, MBP, see Ezekowitz, U.S. Pat.No. 5,270,199) binds to carbohydrates.

MBL is structurally related to the C1q subcomponent of component C1 ofcomplement, and it appears that MBL activates the complement system viaan associated serine protease termed MASP⁴ or p100⁵, which is similar tothe C1r and C1s components of the classical pathway. The new complementactivation pathway is called the MBLectin pathway. According to themechanism postulated for this pathway, MBL binds to specificcarbohydrate structures found on the surface of a range ofmicroorganisms including bacteria, yeast, parasitic protozoa andviruses⁶, and its antimicrobial activity results from activation of theterminal, lytic complement pathway components⁷ or promotingphagocytosis⁸.

Reportedly, the level of MBL in plasma may be geneticallydetermined^(9,10,11). MBL deficiency is associated with susceptibilityto frequent infections with a variety of microorganisms inchildhood^(12,13), and, possibly, in adults^(13,14). Recent informationassociates MBL deficiency with HIV infection and with more rapid deathfollowing development of AIDS^(15,16). MBL binds to the galactosyl formof IgG (GO), which is found at elevated concentrations in rheumatoidarthritis patients, and then activates complement¹⁷. MBL deficiency isalso associated with a predisposition to recurrent spontaneousabortions¹⁸, and also to development of systemic lupus erythrematosus¹⁹.

In the first clinical reconstitution trial, an infant MBL-deficient girlsuffering from recurrent infections was apparently cured by injectionswith purified MBL²⁰. For a recent review on MBL, see ref. 6.

Relatively high frequencies of MBL mutations associated withMBL-deficiency have been reported in all populations studied. Thisobservation has led to the hypothesis that MBL may, in certain cases,render the individual more susceptible to certain intracellularinfectious agents exploiting MBL to gain access to the target tissues²¹.Since MBL is a very powerful activator of the complement system, it mayalso be that inexpedient activation through microbial carbohydrates orendotoxins can lead to damaging inflammatory responses¹⁰. Thus, theoverall survival of a population may benefit from the wide individualrange of MBL concentrations.

MASP-1 (MBP-associated serine protease, MASP) is a serine proteasesimilar in structure to C1r and C1s of the complement pathway althoughit has a histidine loop structure of the type found in trypsin andtrypsin-like serine proteases. MASP-1 has been found to be involved incomplement activation by MBL. A cDNA clone encoding MASP-1 has beenreported that encodes a putative leader peptide of 19 amino acidsfollowed by 680 amino acid residues predicted to form the maturepeptide.

An abstract reports the existence of a second MASP, termed MASP-2.²².

SUMMARY OF THE INVENTION

The invention relates to the isolation and characterization of amannan-binding lectin (MBL) associated serine protease (MASP-2). MASP-2shows some homology with the previously reported MASP (MASP-1) and thetwo C1q-associated serine proteases, C1r and C1s. MBL alone does notprovide a functional MBLectin pathway of complement activation.

We have cloned and sequenced the cDNA encoding MASP-2. In addition, wehave produced anti-MASP-2 antibody and constructed an assay for theestimation of MASP-2 in body fluids or tissue extracts. Furthermore, wehave constructed quantitative assays for the determination of MASP-2activity in serum or plasma, either when present as part of the MBL/MASPcomplex or as free MASP not associated with MBL.

Thus, one aspect of the invention features substantially puremannan-binding lectin associated serine protease-2 (MASP-2) polypeptidesand nucleic acids encoding such polypeptides. Preferably, the MASP-2polypeptide retains one or more MASP-2 functions, such as being capableof associating with mannan-binding lectin (MBL), serine proteaseactivity, or the MASP-2 activity in an in vitro assay for MBLectincomplement pathway function, e.g., in one of the assay systems describedbelow. Some MASP-2 polypeptides according to the invention, e.g., thoseused in binding assays, may be conjugated to a label so as to permitdetection and/or quantification of their presence in the assay. Suitablelabels include enzymes that generate a signal (e.g., visibleabsorption), fluorophores, radionuclides, etc. Other MASP-2 polypeptidesare capable of competitively inhibiting one of the MASP-2 activitiesdescribed above, and thereby are useful in evaluating MASP-2 function.Other MASP-2 polypeptides are useful antigens or haptens for producingantibodies as described below. Compounds which competitively inhibit aMASP-2 activity are also featured. Preferably, such compounds act byinhibiting the serine protease activity of MASP-2 or of a fragment ofMASP-2. Such compounds may include fragments of MBL or of MASP-2 whichcompetitively inhibit the MBL-MASP-2 interactions critical to complementactivation by the MBLectin pathway, as well as compounds, e.g., peptidefragments, which inhibit the catalytic cleavage of complement factors C4and C2 by MASP-2.

Specific polypeptides according to this aspect of the invention include:a) a polypeptide with a molecular mass of 20K and containing thesequence identified as SEQ ID NO:1 [T P L G P K W P E P V F G R L A S PG F P G E Y A N D Q E R R W T L T A P P G Y R]; b) a polypeptide with amolecular mass of 52K and containing the sequence identified as SEQ IDNO:1; c) a polypeptide having the complete amino acid sequence of FIG. 6(SEQ ID NO:2).

Another aspect of the invention includes an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide havingsequence that is at least 85% identical to the sequence of SEQ ID NO:2.

The invention also features isolated nucleic acid sequences encoding theabove mannan-binding lectin associated serine protease-2 (MASP-2)polypeptides. Such nucleic acid sequences may be included in nucleicacid vectors (e.g., expression vectors including those with regulatorynucleic acid elements permitting expression of recombinant nucleic acidin an expression system).

The invention also features antibodies that selectively bind to MASP-2.Such antibodies may be made by any of the well known techniquesincluding polyclonal and monoclonal antibody techniques. The antibodymay be coupled to a compound comprising a detectable marker, so that itcan be used, e.g., in an assay to detect MASP-2.

The polypeptides or antibodies may be formulated into pharmaceuticalcompositions and administered as therapeutics as described below.

The invention also features methods for detecting mannan-binding lectinassociated serine protease-2 (MASP-2). The method comprises; obtaining abiological sample, contacting the biological sample with a MASP-2polypeptide specific binding partner, and detecting the bound complexes,if any, as an indication of the presence of MASP-2 in the biologicalsample. The binding partner used in the assay may be an antibody, or theassay for the MASP-2 may test for complement fixing activity. Theseassays for MASP-2 may also be used for quantitative assays of MASP-2 orMASP-2 activity in biological samples. One of the binding partners maybe specific for MBK thus allowing for the detection of MBL/MASP-2complexes.

Methods for detecting MASP-2 nucleic acid expression are included in theinvention. These methods comprise detecting RNA having a sequenceencoding a MASP-2 polypeptide by mixing the sample with a nucleic acidprobe that specifically hybridizes under stringent conditions to anucleic acid sequence encoding all or a fragment of MASP-2.

The invention also features methods for treating patients deficient inMASP-2 or MASP-2 activity. This is accomplished by administering to thepatient MASP-2 polypeptide or nucleic acid encoding MASP-2. Because itis sometimes desirable to inhibit MASP-2 activity, the inventionincludes a method for inhibiting the activity of MASP-2 by administeringto the patient a compound that inhibits expression or activity ofMASP-2. Inhibition of MASP-2 activity may also be achieved byadministering a MASP-2 anti-sense nucleic acid sequence.

The invention features an assay for polymorphisms in the nucleic acidsequence encoding MASP-2. A method of detecting the presence ofMASP-2-encoding nucleic acid in a sample is claimed. As an example, themethod may include mixing the sample with at least one nucleic acidprobe capable of forming a complex with MASP-2-encoding nucleic acidunder stringent conditions, and determining whether the probe is boundto sample nucleic acid. The invention thus includes nucleic acid probecapable of forming a complex with MASP-2-encoding nucleic acid understringent conditions.

The invention features an assay for polymorphisms in the polypeptidesequence comprising MASP-2 or its precursor.

MASP-2 assays are useful for the determination of MASP-2 levels andMASP-2 activity in patients suffering from various diseases such asinfections, inflammatory diseases and spontaneous recurrent abortion.MASP-2 is useful for the treatment of infections when MASP-2 function issuboptimal, and inhibition of MASP-2 activity is useful for regulationof inflammation and adverse effects caused by activity of the MBLectinpathway.

By “mannan-binding lectin associated serine protease-2” or “MASP-2” ismeant the polypeptide or activity called “mannan-binding proteinassociated serine protease-2” or “mannose-binding protein associatedserine protease” or any other polypeptide having substantial sequenceidentity with SEQ ID NO:2.

The terms “protein” and “polypeptide” are used herein to describe anychain of amino acids, regardless of length or post-translationalmodification (for example, glycosylation or phosphorylation). Thus, theterm “MASP-2 polypeptide” includes full-length, naturally occurringMASP-2 protein, as well as recombinantly or synthetically producedpolypeptide that corresponds to a full-length naturally occurring MASP-2polypeptide, or to particular domains or portions of a naturallyoccurring protein. The term also encompasses mature MASP-2 which has anadded amino-terminal methionine (which is useful for expression inprokaryotic cells).

The term “purified” as used herein refers to a nucleic acid or peptidethat is substantially free of cellular material, viral material, orculture medium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized.

By “isolated nucleic acid molecule” is meant a nucleic acid moleculethat is separated in any way from sequences in the naturally occurringgenome of an organism. Thus, the term “isolated nucleic acid molecule”includes nucleic acid molecules that are not naturally occurring, e.g.,nucleic acid molecules created by recombinant DNA techniques.

The term “nucleic acid molecule” encompasses both RNA and DNA, includingcDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA.Where single-stranded, the nucleic acid may be a sense strand or anantisense strand.

The invention also encompasses nucleic acid molecules that hybridize,preferably under stringent conditions, to a nucleic acid moleculeencoding an MASP-2 polypeptide (e.g., a nucleic acid molecule having thesequence encoding SEQ ID NO:2, e.g., the protein encoding portion of thecDNA sequence shown in FIG. 6—SEQ ID NO:3). In addition, the inventionencompasses nucleic acid molecules that hybridize, preferably understringent conditions, to a nucleic acid molecule having the sequence ofthe MASP-2 encoding cDNA contained in a clone. Preferably thehybridizing nucleic acid molecule consists of 400, more preferably 200nucleotides.

Preferred hybridizing nucleic acid molecules encode an activitypossessed by MASP-2, e.g., they bind MBL and have activity in theMBLectin complement pathway, and can act as serine proteases.

The invention also features substantially pure or isolated MASP-2polypeptides, preferably those that correspond to various functionaldomains of MASP-2, or fragments thereof. The polypeptides of theinvention encompass amino acid sequences that are substantiallyidentical to the amino acid sequence shown in FIG. 6.

The polypeptides of the invention can also be chemically synthesized,synthesized by recombinant technology, or they can be purified fromtissues in which they are naturally expressed, according to standardbiochemical methods of purification.

Also included in the invention are “functional polypeptides” whichpossess one or more of the biological functions or activities of MASP-2.These functions or activities are described in detail in thespecification. A functional polypeptide is also considered within thescope of the invention if it serves as an antigen for production ofantibodies that specifically bind to MASP-2 or fragments (particularlydeterminant containing fragments) thereof.

The functional polypeptides may contain a primary amino acid sequencethat has been modified from those disclosed herein. Preferably thesemodifications consist of conservative amino acid substitutions, asdescribed herein. The polypeptides may be substituted in any mannerdesigned to promote or delay their catabolism (increase theirhalf-life).

Polypeptides or other compounds of interest are said to be“substantially pure” when they are distinct from any naturally occurringcomposition, and suitable for at least one of the uses proposed herein.While preparations that are only slightly altered with respect tonaturally occurring substances may be somewhat useful, more typically,the preparations are at least 10% by weight (dry weight) the compound ofinterest. Preferably, the preparation is at least 60%, more preferablyat least 75%, and most preferably at least 90%, by weight the compoundof interest. Purity can be measured by any appropriate standard method,for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A polypeptide of nucleic acid molecule is “substantially identical” to areference polypeptide or nucleic acid molecule if it has a sequence thatis at least 85%, preferably at least 90%, and more preferably at least95%, 98%, or 99% identical to the sequence of the reference polypeptideor nucleic acid molecule.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide which is 50% identical to the referencepolypeptide over its entire length. Of course, many other polypeptideswill meet the same criteria.

In the case of polypeptide sequences which are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; aspargine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.

For polypeptides, the length of the reference polypeptide sequence willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least amino acids, and most preferably 35amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, thelength of the reference nucleic acid sequence will generally be at least50 nucleotides, preferably at least 60 nucleotides, more preferably atleast 75 nucleotides, and most preferably 100 nucleotides or 300nucleotides.

Sequence identity can be measured using sequence analysis software (forexample, the Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705), with the default parameters as specifiedtherein.

The nucleic acid molecules of the invention can be inserted into avector, as described below, which will facilitate expression of theinsert. The nucleic acid molecules and the polypeptides they encode canbe used directly as diagnostic or therapeutic agents, or can be used(directly in the case of the polypeptide or indirectly in the case of anucleic acid molecule) to generate antibodies that, in turn, areclinically useful as a therapeutic or diagnostic agent. Accordingly,vectors containing the nucleic acid of the invention, cells transfectedwith these vectors, the polypeptides expressed, and antibodiesgenerated, against either the entire polypeptide or an antigenicfragment thereof, are among the preferred embodiments.

The invention also features antibodies, e.g., monoclonal, polyclonal,and engineered antibodies, which specifically bind MASP-2. By“specifically binds” is meant an antibody that recognizes and binds to aparticular antigen, e.g., the MASP-2 polypeptide of the invention, butwhich does not substantially recognize or bind to other molecules in asample, e.g., a biological sample, which includes MASP-2. References toconstructs of antibody (or fragment thereof) coupled to a compoundcomprising a detectable marker includes constructs made by anytechnique, including chemical means or by recombinant techniques.

The invention also features antagonists and agonists of MASP-2 that caninhibit or enhance one or more of the functions or activities of MASP-2,respectively. Suitable antagonists can include small molecules (i.e.,molecules with a molecular weight below about 500), large molecules(i.e., molecules with a molecular weight above about 500), antibodiesthat bind and “neutralize” MASP-2 (as described below), polypeptideswhich compete with a native form of MASP-2 for binding to a protein,e.g., MBL, and nucleic acid molecules that interfere with transcriptionof MASP-2 (for example, antisense nucleic acid molecules and ribozymes).Agonists of MASP-2 also include small and large molecules, andantibodies other than “neutralizing” antibodies.

The invention also features molecules which can increase or decrease theexpression of MASP-2 (e.g., by influencing transcription ortranslation). Small molecules (i.e., molecules with a molecular weightbelow about 500), large molecules (i.e., molecules with a molecularweight above about 500), and nucleic acid molecules that can be used toinhibit the expression of MASP-2 (for example, antisense and ribozymemolecules) or to enhance their expression (for example, expressionconstructs that place nucleic acid sequences encoding MASP-2 under thecontrol of a strong promoter system), and transgenic animals thatexpress a MASP-2 transgene.

The invention encompasses methods for treating disorders associated withaberrant expression or activity of MASP-2. Thus, the invention includesmethods for treating disorders associated with excessive expression oractivity of MASP-2. Such methods entail administering a compound whichdecreases the expression or activity of MASP-2. The invention alsoincludes methods for treating disorders associated with insufficientexpression of MASP-2. These methods entail administering a compoundwhich increases the expression or activity of MASP-2.

By “competitively inhibiting” serine protease activity is meant that,for example, the action of an altered MBL or fragment thereof that canbind to a MASP-2 peptide, reversibly or irreversibly without activatingserine protease activity. Conversely, a fragment of MASP-2, e.g., apolypeptide encompassing the N-terminal part of MASP-2, maycompetitively inhibit the binding of the intact MASP-2 and thuseffectively inhibit the activation of MASP-2.

The invention also features methods for detecting a MASP-2 polypeptide.Such methods include: obtaining a biological sample; contacting thesample with an antibody that specifically binds MASP-2 under conditionswhich permit specific binding; and detecting any antibody-MASP-2complexes formed.

In addition, the present invention encompasses methods and compositionsfor the diagnostic evaluation, typing, and prognosis of disordersassociated with inappropriate expression or activity of MASP-2. Forexample, the nucleic acid molecules of the invention can be used asdiagnostic hybridization probes to detect, for example, inappropriateexpression of MASP-2 or mutations in the MASP-2 gene. Such methods maybe used to classify cells by the level of MASP-2 expression.

Alternatively, the nucleic acid molecules can be used as primers fordiagnostic PCR analysis for the identification of gene mutations,allelic variations and regulatory defects in the MASP-2 gene. Thepresent invention further provides for diagnostic kits for the practiceof such methods.

The invention features methods of identifying compounds that modulatethe expression or activity of MASP-2 by assessing the expression oractivity of MASP-2 in the presence and absence of a selected compound. Adifference in the level of expression or activity of MASP-2 in thepresence and absence of the selected compound indicates that theselected compound is capable of modulating expression or activity orMASP-2. Expression can be assessed either at the level of geneexpression (e.g., by measuring mRNA) or protein expression by techniquesthat are well known to skilled artisans. The activity of MASP-2 can beassessed functionally, i.e., by assaying the ability of the compound toactivate complement.

The preferred methods and materials are described below in exampleswhich are meant to illustrate, not limit, the invention. Skilledartisans will recognize methods and materials that are similar orequivalent to those described herein, and that can be used in thepractice or testing of the present invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-1 b depict a Western blot of human plasma proteins purified bysugar affinity chromatography.

FIG. 2 shows the sequence alignment²¹ of the amino acid sequences ofMASP-2 (clone ph1-4) (SEQ ID NO:2), MASP-1^(17, 22) (SEQ ID NO:6),C1r^(23, 24) (SEQ ID NO:7) and C1s^(25, 26) (SEQ ID NO:8).

FIGS. 3 a 1 to 3 a 3 and 3 b 1 to 3 b 3 are representations of theresults demonstrating molecular complexes formed between MBL, MASP-1 andMASP-2.

FIGS. 4 a-4 b are representations of Western blots demonstrating theactivation of C4 by C1s and MASP-2.

FIG. 5 illustrates the three pathways of complement activation.

FIG. 6 shows the cDNA sequence (SEQ ID NO:3) and deduced amino acidsequence (SEQ ID NO:2) of MASP-2.

DESCRIPTION OF THE PREFERRED EMBODIMENTS MASP-2 Nucleic Acid Molecules

The MASP-2 nucleic acid molecules of the invention can be cDNA, genomicDNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded (i.e., either a sense or an antisense strand). Fragmentsof these molecules are also considered within the scope of theinvention, and can be produced, for example, by the polymerase chainreaction (PCR) or generated by treatment with one or more restrictionendonucleases. A ribonucleic acid (RNA) molecule can be produced by invitro transcription. Preferably, the nucleic acid molecules encodepolypeptides that, regardless of length, are soluble under normalphysiological conditions.

The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide (for example, the polypeptide of SEQ ID NO:2). Inaddition, these nucleic acid molecules are not limited to sequences thatonly encode polypeptides, and thus, can include some or all of thenon-coding sequences that lie upstream or downstream from a codingsequence.

The nucleic acid molecules of the invention can be synthesized (forexample, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. Thus, the nucleic acidscan be those of a human, mouse, rat, guinea pig, cow, sheep, horse, pig,rabbit, monkey, dog, or cat. Combinations or modifications of thenucleotides within these types of nucleic acids are also encompassed.

In addition, the isolated nucleic acid molecules of the inventionencompass fragments that are not found as such in the natural state.Thus, the invention encompasses recombinant molecules, such as those inwhich a nucleic acid molecule (for example, an isolated nucleic acidmolecule encoding MASP-2) is incorporated into a vector (for example, aplasmid or viral vector) or into the genome of a heterologous cell (orthe genome of a homologous cell, at a position other than the naturalchromosomal location). Recombinant nucleic acid molecules and usestherefore are discussed further below.

In the event the nucleic acid molecules of the invention encode or actas antisense molecules, they can be used for example, to regulatetranslation of MASP-2. Techniques associated with detection orregulation of nucleic acid expression are well known to skilled artisansand can be used to diagnose and/or treat disorders associated withMASP-2 activity. These nucleic acid molecules are discussed furtherbelow in the context of their clinical utility.

The invention also encompasses nucleic acid molecules that hybridizeunder stringent conditions to a nucleic acid molecule encoding a MASP-2polypeptide. The cDNA sequence described herein (SEQ ID NO:3) can beused to identify these nucleic acids, which include, for example,nucleic acids that encode homologous polypeptides in other species, andsplice variants of the MASP-2 gene in humans or other mammals.Accordingly, the invention features methods of detecting and isolatingthese nucleic acid molecules. Using these methods, a sample (forexample, a nucleic acid library, such as a cDNA or genomic library) iscontacted (or “screened”) with a MASP-2-specific probe (for example, afragment of SEQ ID NO:3 that is at least 12 nucleotides long). The probewill selectively hybridize to nucleic acids encoding relatedpolypeptides (or to complementary sequences thereof). Because thepolypeptide encoded by MASP-2 is related to other serine proteases, theterm “selectively hybridize” is used to refer to an event in which aprobe binds to nucleic acids encoding MASP-2 (or to complementarysequences thereof) to a detectably greater extent than to nucleic acidsencoding other serine proteases (or to complementary sequences thereof).The probe, which can contain at least 12 (for example, 15, 25, 50, 100,or 200 nucleotides) can be produced using any of several standardmethods (see, for example, Ausubel et al., “Current Protocols inMolecular Biology, Vol. I,” Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., NY, 1989). For example, the probe can be generatedusing PCR amplification methods in which oligonucleotide primers areused to amplify a MASP-2-specific nucleic acid sequence (for example, anucleic acid encoding the N-terminus of mature MASP-2) that can be usedas a probe to screen a nucleic acid library, as described in Example 4below, and thereby detect nucleic acid molecules (within the library)that hybridize to the probe.

One single-stranded nucleic acid is said to hybridize to another if aduplex forms between them. This occurs when one nucleic acid contains asequence that is the reverse and complement of the other (this samearrangement gives rise to the natural interaction between the sense andantisense strands of DNA in the genome and underlies the configurationof the “double helix”). Complete complementarity between the hybridizingregions is not required in order for a duplex to form; it is onlynecessary that the number of paired bases is sufficient to maintain theduplex under the hybridization conditions used.

Typically, hybridization conditions are of low to moderate stringency.These conditions favor specific interactions between completelycomplementary sequences, but allow some non-specific interaction betweenless than perfectly matched sequences to occur as well. Afterhybridization, the nucleic acids can be “washed” under moderate or highconditions of stringency to dissociate duplexes that are bound togetherby some non-specific interaction (the nucleic acids that form theseduplexes are thus not completely complementary).

As is known in the art, the optimal conditions for washing aredetermined empirically, often by gradually increasing the stringency.The parameters that can be changed to affect stringency include,primarily, temperature and salt concentration. In general, the lower thesalt concentration and the higher the temperature, the higher thestringency. Washing can be initiated at a low temperature (for example,room temperature) using a solution containing a salt concentration thatis equivalent to or lower than that of the hybridization solution.Subsequent washing can be carried out using progressively warmersolutions having the same salt concentration. As alternatives, the saltconcentration can be lowered and the temperature maintained in thewashing step, or the salt concentration can be lowered and thetemperature increased. Additional parameters can also be altered. Forexample, use of a destabilizing agent, such as formamide, alters thestringency conditions.

In reactions where nucleic acids are hybridized, the conditions used toachieve a given level of stringency will vary. There is not one set ofconditions, for example, that will allow duplexes to form between allnucleic acids that are 85% identical to one another; hybridization alsodepends on unique features of each nucleic acid. The length of thesequence, the composition of the sequence (for example, the content ofpurine-like nucleotides versus the content of pyrimidine-likenucleotides) and the type of nucleic acid (for example, DNA or RNA)affect hybridization. An additional consideration is whether one of thenucleic acids is immobilized (for example, on a filter).

An example of a progression from lower to higher stringency conditionsis the following, where the salt content is given as the relativeabundance of SSC (a salt solution containing sodium chloride and sodiumcitrate; 2×SSC is 10-fold more concentrated than 0.2×SSC). Nucleic acidsare hybridized at 42° C. in 2×SSC/0.1% SDS (sodium dodecylsulfate; adetergent) and then washed in 0.2×SSC/0.1% SDS at room temperature (forconditions of low stringency); 0.2×SSC/0.1% SDS at 42° C. (forconditions of moderate stringency); and 0.1×SSC at 68° C. (forconditions of high stringency). Washing can be carried out using onlyone of the conditions given, or each of the conditions can be used (forexample, washing for 10-15 minutes each in the order listed above). Anyor all of the washes can be repeated. As mentioned above, optimalconditions will vary and can be determined empirically.

A second set of conditions that are considered “stringent conditions”are those in which hybridization is carried out at 50° C. in Churchbuffer (7% SDS, 0.5% NaHPO₄, 1 M EDTA, 1% bovine serum albumin) andwashing is carried out at 50° C. in 2×SSC.

Once detected, the nucleic acid molecules can be isolated by any of anumber of standard techniques (see, for example, Sambrook et al.,“Molecular Cloning, A Laboratory Manual,” 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

The invention also encompasses: (a) expression vectors that contain anyof the foregoing MASP-2-related coding sequences and/or theircomplements (that is, “antisense” sequence); (b) expression vectors thatcontain any of the foregoing MASP-2-related coding sequences operativelyassociated with a regulatory element (examples of which are given below)that directs the expression of the coding sequences; (c) expressionvectors containing, in addition to sequences encoding a MASP-2polypeptide, nucleic acid sequences that are unrelated to nucleic acidsequences encoding MASP-2, such as molecules encoding a reporter ormarker; and (d) genetically engineered host cells that contain any ofthe foregoing expression vectors and thereby express the nucleic acidmolecules of the invention in the host cell.

Recombinant nucleic acid molecule can contain a sequence encoding asoluble MASP-2, mature MASP-2, MASP-2 having a signal sequence, orfunctional domains of MASP-2 such as the serine protease domain, EGFdomain, or the MBL-binding domain. The full length MASP-2 polypeptide, adomain of MASP-2, or a fragment thereof may be fused to additionalpolypeptides, as described below. Similarly, the nucleic acid moleculesof the invention can encode the mature form of MASP-2 or a form thatencodes a polypeptide which facilitates secretion. In the latterinstance, the polypeptide is typically referred to as a proprotein,which can be converted into an active form by removal of the signalsequence, for example, within the host cell. Proproteins can beconverted into the active form of the protein by removal of theinactivating sequence.

The regulatory elements referred to above include, but are not limitedto, inducible and non-inducible promoters, enhancers, operators andother elements, which are known to those skilled in the art, and whichdrive or otherwise regulate gene expression. Such regulatory elementsinclude but are not limited to the cytomegalovirus hCMV immediate earlygene, the early or late promoters of SV40 adenovirus, the lac system,the trp system, the TAC system, the TRC system, the major operator andpromoter regions of phage A, the control regions of fd coat protein, thepromoter for 3-phosphoglycerate kinase, the promoters of acidphosphatase, and the promoters of the yeast α-mating factors.

Similarly, the nucleic acid can form part of a hybrid gene encodingadditional polypeptide sequences, for example, sequences that functionas a marker or reporter. Examples of marker or reporter genes includeβ-lactamase, chloramphenicol acetyltransferase (CAT), adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo^(r), G418^(r)),dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH),thymidine kinase (TK), lacZ (encoding (β-galactosidase), greenfluorescent protein (GFP), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example, ofadditional sequences that can serve the function of a marker orreporter. Generally, the hybrid polypeptide will include a first portionand a second portion; the first portion being a MASP-2 polypeptide andthe second portion being, for example, the reporter described above oran immunoglobulin constant region.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (forexample, E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining the nucleic acid molecules of the invention; yeast (forexample, Saccharomyces and Pichia) transformed with recombinant yeastexpression vectors containing the nucleic acid molecules of theinvention (preferably containing the nucleic acid sequence of MASP-2(SEQ ID NO:3)); insect cell systems infected with recombinant virusexpression vectors (for example, baculovirus) containing the nucleicacid molecules of the invention; plant cell systems infected withrecombinant virus expression vectors (for example, cauliflower mosaicvirus (CaMV) and tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (for example, Ti plasmid)containing MASP-2 nucleotide sequences; or mammalian cell systems (forexample, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (for example, the metallothioneinpromoter) or from mammalian viruses (for example, the adenovirus latepromoter and the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions containing MASP-2 polypeptides or for raising antibodies tothose polypeptides, vectors that are capable of directing the expressionof high levels of fusion protein products that are readily purified maybe desirable. Such vectors include, but are not limited to, the E. coliexpression vector pUR278 (Ruther et al., EMBO J. 2:1791, 1983), in whichthe coding sequence of the insert may be ligated individually into thevector in frame with the lacZ coding region so that a fusion protein isproduced; pIN vectors (Inouye and Inouye, Nucleic Acids Res.13:3101-3109, 1985; Van Heeke and Schuster, J. Biol. Chem.264:5503-5509, 1989); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) can be used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The coding sequence of the insertmay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofthe coding sequence will result in inactivation of the polyhedrin geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed (for example, see Smith et al.,J. Virol. 46:584, 1983; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the nucleic acid molecule of the invention may be ligated to anadenovirus transcription/translation control complex, for example, thelate promoter and tripartite leader sequence. This chimeric gene maythen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(for example, region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a MASP-2 gene product in infectedhosts (for example, see Logan and Shenk, Proc. Natl. Acad. Sci. USA81:3655-3659, 1984). Specific initiation signals may also be requiredfor efficient translation of inserted nucleic acid molecules. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire gene or cDNA, including its own initiation codonand adjacent sequences, is inserted into the appropriate expressionvector, no additional translational control signals may be needed.However, in cases where only a portion of the coding sequence isinserted, exogenous translational control signals, including, perhaps,the ATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:516-544, 1987).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (forexample, glycosylation) and processing (for example, cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Themammalian cell types listed above are among those that could serve assuitable host cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe MASP-2 sequences described above may be engineered. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (for example, promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then switched to a selective media. The selectable marker inthe recombinant plasmid confers resistance to the selection and allowscells to stably integrate the plasmid into their chromosomes and grow toform foci which in turn can be cloned and expanded into cell lines. Thismethod can advantageously be used to engineer cell lines which expressMASP-2. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the gene product and for production of MASP-2 fortherapeutic uses. These methods may also be used to modify cells thatare introduced into a host organism either for experimental ortherapeutic purposes. The introduced cells may be transient or permanentwithin the host organism.

A number of selection systems can be used. For example, the herpessimplex virus thymidine kinase (Wigler, et al., Cell 11:223, 1977),hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski,Proc. Natl. Acad. Sci. USA 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Nail. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA78:2072, 1981); neo, which confers resistance to the aminoglycosideG-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro,which confers resistance to hygromycin (Santerre et al., Gene 30:147,1984).

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Proc. Natl. Acad. Sci. USA 88:8972-8976, 1991). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the gene's open reading frame is translationally fused to anamino-terminal tag consisting of six histidine residues. Extracts fromcells infected with recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose columns and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

MASP-2 Polypeptides

The MASP-2 polypeptides described herein are those encoded by any of thenucleic acid molecules described above and include MASP-2 fragments,mutants, truncated forms, and fusion proteins. These polypeptides can beprepared for a variety of uses, including but not limited to thegeneration of antibodies, as reagents in diagnostic assays, for theidentification of other cellular gene products or compounds that canmodulate the MBLectin response, and as pharmaceutical reagents usefulfor the treatment of inflammation and certain disorders (describedbelow) that are associated with activity of the MBLectin pathway.Preferred polypeptides are substantially pure MASP-2 polypeptides,including those that correspond to the polypeptide with an intact signalsequence (extending from amino acids 1-15 of SEQ ID NO:2), the matureform of the polypeptide (extending from amino acids 16-686 of SEQ IDNO:2) of the human MASP-2 polypeptide as well as polypeptidesrepresenting a part of the MASP-2 polypeptide. Especially preferred arepolypeptides that are soluble under normal physiological conditions.

The invention also encompasses polypeptides that are functionallyequivalent to MASP-2. These polypeptides are equivalent to MASP-2 inthat they are capable of carrying out one or more of the functions ofMASP-2 in a biological system. Preferred MASP-2 polypeptides have 20%,40%, 50%, 75%, 80%, or even 90% of the activity of the full-length,mature human form of MASP-2 described herein. Such comparisons aregenerally based on an assay of biological activity in which equalconcentrations of the polypeptides are used and compared. The comparisoncan also be based on the amount of the polypeptide required to reach 50%of the maximal activity obtainable.

Functionally equivalent proteins can be those, for example, that containadditional or substituted amino acid residues. Substitutions may be madeon the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. Amino acids that are typically considered to providea conservative substitution for one another are specified in the summaryof the invention. D-amino acids may be introduced in order to modify thehalf-life of the polypeptide.

Polypeptides that are functionally equivalent to MASP-2 (SEQ ID NO:2)can be made using random mutagenesis techniques well known to thoseskilled in the art (and the resulting mutant MASP-2 proteins can betested for activity). It is more likely, however, that such polypeptideswill be generated by site-directed mutagenesis (again using techniqueswell known to those skilled in the art). These polypeptides may have anincreased function, i.e., a greater ability to activate the MBLectinpathway. Such polypeptides can be used to enhance the activity ofMBLectin pathway immune function.

To design functionally equivalent polypeptides, it is useful todistinguish between conserved positions and variable positions. This canbe done by aligning the sequence of MASP-2 cDNAs that were obtained fromvarious organisms. Skilled artisans will recognize that conserved aminoacid residues are more likely to be necessary for preservation offunction. Thus, it is preferable that conserved residues are notaltered.

Mutations within the MASP-2 coding sequence can be made to generateMASP-2 peptides that are better suited for expression in a selected hostcell. Introduction of a glycosylation sequence can also be used togenerate a MASP-2 polypeptide with altered biological characteristics.

The invention also features methods for assay of polymorphisms withinthe polypeptide sequence comprising MASP-2 or its precursor. This may beaccomplished by a number of techniques. For example, the purifiedpolypeptide is subjected to tryptic digestion and the resultingfragments analyzed by either one- or two dimensional electrophoresis.The results from analysis of a sample polypeptide are compared to theresults using a known sequence. Also the analysis may encompassseparation of a biological sample (e.g., serum or other body fluids) byeither one- or two-dimensional electrophoresis followed by transfer ofthe separated proteins onto a membrane (Western blot). The membrane isthen reacted with antibodies against MASP-2, followed by a secondarylabelled antibody. The staining pattern is compared with that obtainedusing a sample with a known sequence of modification.

The polypeptides of the invention can be expressed fused to anotherpolypeptide, for example, a marker polypeptide or fusion partner. Forexample, the polypeptide can be fused to a hexa-histidine tag tofacilitate purification of bacterially expressed protein or ahemagglutinin tag to facilitate purification of protein expressed ineukaryotic cells. The MASP-2 polypeptide of the invention, or a portionthereof, can also be altered so that it has a longer circulatinghalf-life by fusion to an immunoglobulin Fc domain (Capon et al., Nature337:525-531, 1989). Similarly, a dimeric form of the MASP-2 polypeptidecan be produced, which has increased stability in vivo.

The polypeptides of the invention can be chemically synthesized (forexample, see Creighton, “Proteins: Structures and Molecular Principles,”W.H. Freeman & Co., NY, 1983), or, perhaps more advantageously, producedby recombinant DNA technology as described herein. For additionalguidance, skilled artisans may consult Ausubel et al. (supra), Sambrooket al. (“Molecular Cloning, A Laboratory Manual,” Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1989), and particularly for examples ofchemical synthesis Gait, M. J. Ed. (“Oligonucleotide Synthesis,” IRLPress, Oxford, 1984).

The invention also features polypeptides that interact with MASP-2 (andthe genes that encode them) and thereby alter the function of MASP-2interacting polypeptides can be identified using methods known to thoseskilled in the art. One suitable method is the “two-hybrid system,”which detects protein interactions in vivo (Chien et al., Proc. Natl.Acad. Sci. USA 88:9578, 1991). A kit for practicing this method isavailable from Clontech (Palo Alto, Calif.).

Anti-MASP-2 Antibodies

Human MASP-2 polypeptides (or immunogenic fragments or analogs) can beused to raise antibodies useful in the invention; such polypeptides canbe produced by recombinant techniques or synthesized (see, for example,“Solid Phase Peptide Synthesis,” supra; Ausubel et al., supra). Ingeneral, the peptides can be coupled to a carrier protein, such as KLH,as described in Ausubel et al., supra, mixed with an adjuvant, andinjected into a host mammal. Also the carrier could be PPD. Antibodiescan be purified by peptide antigen affinity chromatography.

In particular, various host animals can be immunized by injection with aMASP-2 protein or polypeptide. Host animals include rabbits, mice,guinea pigs, rats, and chickens. Various adjuvants that can be used toincrease the immunological response depend on the host species andinclude Freund's adjuvant (complete and incomplete), mineral gels suchas aluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Potentially useful human adjuvantsinclude BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.Polyclonal antibodies are heterogeneous populations of antibodymolecules that are contained in the sera of the immunized animals.

Antibodies within the invention therefore include polyclonal antibodiesand, in addition, monoclonal antibodies, humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,and molecules produced using a Fab expression library, and antibodies orfragments produced by phage display techniques.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be prepared using the MASP-2 proteinsdescribed above and standard hybridoma technology (see, for example,Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol.6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling etal., “Monoclonal Antibodies and T Cell Hybridomas,” Elsevier, NY, 1981;Ausubel et al., supra).

In particular, monoclonal antibodies can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture such as described in Kohler et al., Nature256:495, 1975, and U.S. Pat. No. 4,376,110; the human B-cell hybridomatechnique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al.,Proc. Natl. Acad. Sci. USA 80:2026, 1983), and the EBV-hybridomatechnique (Cole et al., “Monoclonal Antibodies and Cancer Therapy,” AlanR. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. (In the case of chickens, the immunoglobulin class can also beIgY.) The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. The ability to produce high titers ofmAbs in vivo makes this the presently preferred method of production,but in some cases, in vitro production will be preferred to avoidintroducing cancer cells into live animals, for example, in cases wherethe presence of normal immunoglobulins coming from the acitis fluids areunwanted, or in cases involving ethical considerations.

Once produced, polyclonal, monoclonal, or phage-derived antibodies aretested for specific MASP-2 recognition by Western blot orimmunoprecipitation analysis by standard methods, e.g., as described inAusubel et al., supra. Antibodies that specifically recognize and bindto MASP-2 are useful in the invention. For example, such antibodies canbe used in an immunoassay to monitor the level of MASP-2 produced by ananimal (for example, to determine the amount or subcellular location ofMASP-2).

Preferably, antibodies of the invention are produced using fragments ofthe MASP-2 protein which lie outside highly conserved regions and appearlikely to be antigenic, by criteria such as high frequency of chargedresidues. In one specific example, such fragments are generated bystandard techniques of PCR, and are then cloned into the pGEX expressionvector (Ausubel et al., supra). Fusion proteins are expressed in E. coliand purified using a glutathione agarose affinity matrix as described inAusubel et al., supra.

In some cases it may be desirable to minimize the potential problems oflow affinity or specificity of antisera. In such circumstances, two orthree fusions can be generated for each protein, and each fusion can beinjected into at least two rabbits. Antisera can be raised by injectionsin a series, preferably including at least three booster injections.

Antisera is also checked for its ability to immunoprecipitaterecombinant MASP-2 proteins or control proteins, such as glucocorticoidreceptor, CAT, or luciferase.

The antibodies can be used, for example, in the detection of the MASP-2in a biological sample as part of a diagnostic assay. Antibodies alsocan be used in a screening assay to measure the effect of a candidatecompound on expression or localization of MASP-2. Additionally, suchantibodies can be used in conjunction with the gene therapy techniquesdescribed to, for example, evaluate the normal and/or engineeredMASP-2-expressing cells prior to their introduction into the patient.Such antibodies additionally can be used in a method for inhibitingabnormal MASP-2 activity.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851, 1984;Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452,1984) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can beadapted to produce single chain antibodies against a MASP-2 protein orpolypeptide. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science, 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to MASP-2 can, in turn, be used to generate anti-idiotypeantibodies that resemble a portion of MASP-2 using techniques well knownto those skilled in the art (see, e.g., Greenspan et al., FASEB J.7:437, 1993; Nissinoff, J. Immunol. 147:2429, 1991). For example,antibodies that bind to MASP-2 and competitively inhibit the binding ofa ligand of MASP-2 can be used to generate anti-idiotypes that resemblea ligand binding domain of MASP-2 and, therefore, bind and neutralize aligand of MASP-2 such as MBL. Such neutralizing anti-idiotypicantibodies or Fab fragments of such anti-idiotypic antibodies can beused in therapeutic regimens.

Antibodies can be humanized by methods known in the art. For example,monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals are also features of the invention (Green et al., NatureGenetics 7:13-21, 1994; see also U.S. Pat. Nos. 5,545,806 and 5,569,825,both of which are hereby incorporated by reference).

The methods described herein in which anti-MASP-2 antibodies areemployed may be performed, for example, by utilizing pre-packageddiagnostic kits comprising at least one specific MASP-2 nucleotidesequence or antibody reagent described herein, which may be convenientlyused, for example, in clinical settings, to diagnose patients exhibitingsymptoms of the disorders described below.

Quantitative Assays of MASP-2

As an example only, quantitative assays may be devised for theestimation of MASP-2 concentrations in body fluids or organ (biopsy)extracts. Such assays may be fluid phase or solid phase. Examples arecompetitive and non-competitive ELISAs. As an example of the latter,microtiter wells are coated with anti-MASP-2 antibody, incubated withsamples, and the presence of MASP-2 visualized with enzyme-labelledantibody followed by substrate that deposits a colored compound.Alternatively, a label such as europium may be used and the detectionmade by use of time resolved fluorometry.

Assays of the functional activity of MASP-2, either alone or as part ofthe MBL/MASP complex may be performed by several methods. As an exampleof a test for MBL/MASP-2 complex, the test sample is applied ontomannan-coated micro wells and C4 is added to estimate the C4-cleavingactivity, or C3 is added to estimate the C3 cleaving activity of thegenerated C3 convertase. Assay of MASP-2 not occurring as part of theMBL/MASP complex is carried out similarly, but MBL is added either tothe micro well or to the sample before adding this to the mannan-coatedwell. Before the addition of MBL the sample may be depleted of MBL andMBL/MASP-1 and MBL/MASP-2 complexes by treatment with solid phasemannan, e.g. attached to beads, or by solid phase anti-MBL antibodies,or by treatment with a suitable concentration of a precipitating agent,e.g., PEG, which precipitates the complex but leaves MASP-2 in thesupernatant. The assay is carried out at conditions which minimize oreliminate interference from the classical complement activation pathwayand the alternative complement activation pathway.

Assays estimating the activity of MASP-2 or MASP-2 may be used fordiagnostic and treatment purposes in samples from individuals, notablythose suffering from infectious or inflammatory disease.

MASP-2 for Therapy

Therapeutic use of components specified in the claims may be applied insituations where a constitutional or temporary deficiency in MASP-2renders the individual susceptible to one or more infections, orsituations where the individual cannot neutralize an establishedinfection. MASP-2 or MBL/MASP complexes can be administered, preferablyby intravenous infusions, in order to improve the individual's immunedefense.

We believe MASP-2 is required for the powerful antimicrobial activity ofthe MBL/MASP complex, and deficiency in MASP-2, either geneticallydetermined or acquired, will therefore compromise an individual'sresistance to infections and ability to combat established infections.Reconstitution with natural or recombinant MASP-2 is a useful treatmentmodality in such situations. Recombinant MASP-2 may be in the form ofthe whole molecule, parts of the molecule, or the whole or part thereofattached by any means to another structure in order to modulate theactivity. The recombinant products may be identical in structure to thenatural molecule or slightly modified to yield enhanced activity ordecreased activity when such is desired.

Reconstitution therapy with MBL, either natural or recombinant, requiresthat the recipient has sufficient MASP-2 for the expression of MBL/MASPactivity. Thus, MASP-2 must be included in the therapeutic preparationwhen the patient has insufficient MASP-2 activity.

Assays for MASP-2

Therapy with MASP-2 (or MASP-2 inhibitors) must usually be preceded bythe estimation of MASP-2 in serum or plasma from the patient. Examplesof such assays are described below.

Assays for MASP-2 Antigen

MASP-2 protein is conveniently estimated as antigen using one of thestandard immunological procedures.

As an example only, a quantitative TRIFMA (time resolvedimmunofluorometric assay) for MASP-2 was constructed by 1) coatingmicrotiter wells with 1 μg anti-C′ MASP-2 antibody; 2) blocking withTween-20; 3) applying test samples, e.g. diluted plasma or serumsamples; 4) applying Eu-labelled anti-N′ MASP-2 antibody; 5) applyingenhancement solution (Wallac Ltd); 6) reading the Eu on a time resolvedfluorometer. (Estimation by ELISA may be carried out similarly, e.g. byusing biotin-labelled anti-N′ MASP-2 in step 4; alkalinephosphatase-labelled avidin in step 5; 6) apply substrate; and 7) readthe colour intensity.) Between each step, the plate was incubated atroom temperature and washed, except between step 6 and 7. A calibrationcurve may be constructed using dilutions of pooled normal plasma,arbitrarily said to contain 1 unit of MASP-2 per ml. The antibodies usedin this first version of a MASP-2 assay were raised against syntheticpeptides and reacted poorly with native MASP-2. The samples are thuspretreated with SDS on a boiling water bath for 5 min. and the SDSneutralized with non-ionic detergent (Triton X-100) before the assay. Afurther development of the assay employs antibodies reacting with nativeMASP-2, thus rendering the SDS treatment superfluous.

Assays may be similarly constructed using antibodies, polyclonal ormonoclonal or recombinant antibodies, which reacts with MASP-2, naturalor recombinant, or parts thereof.

Through the use of antibodies reacting selectively with intact MASP-2 orwith activation products, or through combination of antibodies againstvarious parts of the molecule, assays may be constructed for theestimation of the activation in vivo of the MBLectin pathway. Theseassays will be useful for the determination of inflammation caused bythe activation of this pathway.

Assays for MASP-2 Activity of the MBL/MASP Complex

MASP-2 may be estimated by its capacity to activate the complementsystem.

When C4 is cleaved by MASP-2 an active thiol ester is exposed and C4becomes covalently attached to nearby nucleophilic groups. A substantialpart of the C4b will thus become attached to the coated plastic well andmay be detected by anti-C4 antibody. A quantitative TRIFMA for MASP-2activity was constructed by 1) coating microtiter wells with 1 μg mannanin 100 μl buffer; 2) blocking with Tween-20; 3) applying test samples,e.g. diluted plasma or serum samples; 4) applying purified complementfactor C4 at g/ml; 5) incubate for one hour at 37° C.; 6) applyingEu-labelled anti-C4 antibody; 7) applying enhancement solution; and 8)reading the Eu by time resolved fluorometry. (Estimation by ELISA may becarried out similarly, e.g. by applying biotin-labelled anti-C4 in step6; 7) apply alkaline phosphatase-labelled avidin; 8) apply substrate;and 9) read the colour intensity). Between each step the plate wasincubated at room temperature and washed, except between step 7 and 8. Acalibration curve can be constructed using dilutions of one selectednormal plasma, arbitrarily said to contain 1 unit of MBL/MASP-2 activityper ml. The assay is carried out at conditions which preclude activationof C4 by the classical or alternative complement activation pathways.The activation of C4 was completely inhibited by the serine proteaseinhibitor benzamidine. Activation of the classical pathway iseffectively eliminated by carrying out step 3) in the presence ofsufficiently high ionic strength (0.7 to 2.0 NaCl; preferably about 1.0M NaCl) which does not interfere with the MBL/MASP complex butcompletely destroys the C1qrs ecomplex; activation of the alternativepathway is effectively precluded by assaying at a dilution of 20-fold orgreater.

Assays for the Estimation of Free MASP-2 Activity

The estimation of MASP-2 activity in samples from MBL-deficientindividuals is carried out on wells coated with MASP-free MBL. Theestimation of free MASP in samples from individuals with MBL is carriedout by first removing MBL/MASP-1 and MBL/MASP-2 complexes by incubatingwith Sepharose-coupled mannan (300 μl of fold diluted plasma or serum isincubated with 10 μl beads), and then analyzing the supernatant.

The assay carried out in the TRIFMA formate proceeds as follows: 1)coating microtiter wells with 1 g mannan in 100 μl buffer; 2) blockingwith Tween-20; 3) incubate sample at a 1000 fold dilution in buffer with100 ng of MASP-free MBL/ml, and applying 100 μl of the mixture per well;4) incubate over night at 4° C.; 4) wash and applying purifiedcomplement factor C4 at 5 μg/ml; 5) incubate for one hour at 37° C.; 6)applying Eu-labelled anti-C4 antibody; 7) applying enhancement solution;and 8) reading the Eu by time resolved fluorometry. (Estimation by ELISAmay be carried out similarly, e.g. by applying biotin-labeled anti-C4 instep 6; 7) apply alkaline phosphatase-labelled avidin; 8) applysubstrate; and 9) read the color intensity.) Between each step the platewas washed, except between step 7 and 8. A calibration curve may beconstructed using dilutions of one selected MBL-deficient plasma,arbitrarily said to contain 1 unit of MASP-2 activity per ml. The assayis carried out at conditions which preclude activation of C4 by theclassical or alternative complement activation pathways (see above).

Inhibition of MASP-2 Activity

Inhibitors of the biological activity of MASP-2 may be employed tocontrol the complement activating activity and inflammatory activity ofMASP-2. Such inhibitors may be substrate analogues representing targetstructures of C2 or C4. Inhibitors may be of peptide nature, modifiedpeptides, or any organic molecule that inhibits the activity of MASP-2competitively or non-competitively. The inhibitor may be modified tostay in circulation for short or longer time, and constructed to begiven by injection or perorally. Inhibitors may be fragments of MASP-2,produced from natural or recombinant MASP-2, by chemical or enzymaticprocedures. Inhibitors may be naturally occurring shorter forms ofMASP-2. Inhibitors may be in soluble form or coupled to a solid phase. Asolid phase could be a compatible surface such as used in extracorporealblood or plasma flow devices.

Microbial carbohydrates or endogenous oligosaccharides may provokeundesirable activation of the MBL/MASP complex resulting in damaginginflammatory responses. This pathophysiological activity may be reducedthough the administration of inhibitors of MASP-2 activity such asPefabloc. Also other enzyme inhibitors (PMSF, benzamidine, etc.) haveproved effective when assayed in the TRIFMA for MASP-2 activity.Obviously, when designing inhibitors for in vivo use toxicity is a majorconsideration, and highly specific inhibitors can be assumed to be lesstoxic than more broadly reactive inhibitors. Specific inhibitors may begenerated through using peptides, peptide analogues or peptidederivatives representing the target structures on complement factor C4or C2 molecules. Another type of inhibitors may be based on antibodies(or fragments of antibodies) against the active site of MASP-2 or otherstructures on MASP-2 thus inhibiting the activity of MASP-2. Inhibitorsmay also be directed towards inhibition of the activation of MASP-2,thought to be effected by MASP-1, i.e. the target structure for MASP-1on MASP-2 would be a suitable inhibitor of this type. Another type ofinhibitor would prevent the binding of MASP-2 to MBL and thereby theactivation of MASP-2. The N-terminal 20 kDa fragment of MASP-2 may be asuitable inhibitor of this type. More specifically one can localize theprecise part of the polypeptide chain which mediates the binding ofMASP-2 to MBL and use the synthetic peptide or analogous structures asinhibitor. Inhibitors may be substituted with D amino acids for L-aminoacids.

Also, inhibitors could be RNA or single stranded DNA isolated by SELEX(systemic evolution of ligands by exponential enrichment) using MASP-2or fragments thereof as selecting molecule. The leader sequence ofMASP-2 is shown elsewhere in this application.

MASP-2 activity may be controlled by the conversion of the pro-enzymeform of MASP-2 into activated MASP-2 through the action of MASP-1 or anyother substance simulating the activity of MASP-1.

EXAMPLES Example 1 Identification of MASP-2

Human plasma proteins and protein complexes, that bind to carbohydratesin a calcium-dependent manner (i.e. lectins and lectin-associatedproteins), were purified by affinity chromatography on mannan- andN-acetylglucosamine-derivatized Sepharose beads. Pooled CPD-plasma (2.5l), diluted with buffer containing EDTA and enzyme inhibitors werepassed through Sepharose 2B CL and mannan-Sepharose. A thrombininhibitor, PPACK (D-phenylalanyl-prolyl-arginyl-chloromethyl ketone) andCaCl₂ were added. The pool was passed through Sepharose 2B-CL andmannan-Sepharose, and the proteins binding calcium-dependently tomannan-Sepharose were eluted with EDTA-containing buffer. The eluate wasrecalcified, passed through a GlcNAc-Sepharose column which was elutedas above to yield 20 ml “lectin preparation”.

This protein preparation was analyzed by SDS-PAGE and blotting onto aPVDF-membrane. Development of the blot with chicken antibody raisedagainst a bovine lectin preparation²⁵ revealed a protein with an M_(r)of 52 kDa as well as MBL at 32 kDa. The 52 kDa band was subjected toNH₂-terminal amino acid sequence analysis. The sequence showedsimilarity to that of the previously described MASP (MASP-1). Antibodyraised against a synthetic peptide representing the 19 NH₂-terminalamino acids (anti N′ MASP-2 antiserum) recognized the 52 kDa molecule aswell as a molecule with a mobility corresponding to 20 kDa (FIG. 1, lane1). Under non-reducing conditions, a polypeptide of 76 kDa was detectedusing the anti-N′-MASP-2 antiserum (FIG. 1, lane 2), indicating thepresence of intra-chain disulphide bonds. The 20 kDa polypeptide wasfound to have the same NH₂-terminal sequence as the 52 kDa polypeptideand is likely to represent a truncated form of the latter. The directlydetermined amino acid sequences (NH₂-terminal as well as those ofinternal peptides) are indicated in FIG. 6. Two dimensional SDS-PAGEwith the first dimension under nonreducing conditions and the seconddimension under reducing conditions showed the 52 kDa polypeptide to bea part of a disulphide-linked protein with an M_(r) of 76 kDa. Apolypeptide of 31 kDa (FIG. 1, lane 3), likely to represent theremaining part of the protein, was also recognized as part of the 76 kDaprotein by an antiserum (anti-C′ MASP-2) raised against syntheticpeptides representing sequences in the COOH-terminal part of the protein(determined by cDNA sequencing techniques; see below). The 76 kDa bandseen with the anti-N′ MASP-2 antibody under non-reducing conditions wasalso recognized by the anti-C′ MASP-2 antibody (FIG. 1, lane 4).

FIG. 1 b depicts SDS-PAGE in two dimensions, the first dimension undernon-reducing conditions. The lane was cut out, incubated in samplebuffer containing dithiothreitol (DTT), placed on top of anotherSDS-PAGE gel, and after electrophoresis, the gel was blotted and theblot developed with anti-N′ MASP-2 antibody. The positions of molecularweight markers are indicated.

Example 2 Preparation of Antibodies Against Mannan-Binding LectinAssociated Serine Proteases

Animals, primed with BCG (Bacillus Calmette Guérin vaccine) wereimmunized with synthetic peptides coupled to PPD (tuberculin purifiedprotein derivative) according to C. Koch, The State Serum Institute,Copenhagen. Antibody designated anti-N′ MASP-1, anti-C′ MASP-1 andanti-N′ MASP-2 were from rabbits immunized with peptides correspondingto the first 19 amino acid residues of MASP-1 (SEQ ID NO:6), the last 19amino acid residues of MASP-1 (SEQ ID NO:6), and the first 19 amino acidresidues of MASP-2 (SEQ ID NO:2), respectively. Chicken anti-C′ MASP-2antibody was from chickens immunized with a mixture of two peptidesrepresenting sequences in the C-terminal part of MASP-2 (residues 505 to523 and 538 to 556). All peptides had an additional C-terminal cysteinefor coupling. Antibody and normal chicken IgG was purified from yolk²⁶.Monoclonal anti-MBL antibody, IgG₁-kappa (clone 131-1) and controlIgG₁-kappa (clone MOPC 21) were purified by Protein A affinitychromatography. F(ab′)₂ rabbit anti-human C4 and F(ab′)₂ rabbitanti-human C1q were produced by pepsin digestion of rabbit anti-human C4and rabbit anti-human C1q (DAKO, Glostrup, Denmark). For staining ofWestern blots antibodies were used at 1 g/ml. Bound chicken antibody wasvisualized with rabbit anti-chicken IgG followed by peroxidase-labelledgoat anti-rabbit IgG and development using the enhancedchemiluminescence technique. Bound mouse and rabbit antibodies werevisualized with peroxidase-labelled rabbit anti-mouse IgG andperoxidase-labelled goat anti-rabbit IgG, respectively.

Example 3 Amino Acid Sequencing of the 52 kDa and the 20 kDaPolypeptides

The lectin preparation was concentrated, subjected to SDS-PAGE, andtransferred to a PVDF membrane. Two strips were developed withanti-bovine lectin antibody²⁵. The rest of the blot was stained withCoomassie Brilliant Blue. The band corresponding to the immuno-stained52 kDa band, judged to represent about 5% of the total Coomassie-stainedproteins, was cut out and subjected to sequencing on an AppliedBiosystems protein sequencer. After production of anti-N′ MASP-2antibody, a similar Western blot was performed using the anti-N-MASP-2antibody. The NH₂-termini of the proteins in the 52 kDa and the 20 kDabands visualized with this antibody were sequenced. Peptides wereprepared by trypsin digestion of the proteins in the two bands fromanother blot, fractionated by reverse phase chromatography and thepeptides in the major peaks were subjected to sequencing.

Example 4 Cloning and Sequencing of MASP-2

The liver is the primary site of synthesis of C1r, C1s, and MASP-1.Thus, RNA from liver was used as template for RT-PCR with primersdeduced from the obtained peptide sequences. First strand synthesis ofcDNA was carried out with 1.3 μg human liver RNA using a First-StrandcDNA Synthesis Kit (Pharmacia). PCR was performed on this cDNA usingdegenerate sense and antisense primers derived from the amino acidsequences EYANDQER (SEQ ID NO:4) and KPFTGFEA (SEQ ID NO:5),respectively. The PCR program consisted of 1 cycle with annealing at 50°C.; 1 cycle with annealing at 55° C., and 33 cycles with annealing at60° C. The resulting 300 bp PCR product was cloned into the E. coliplasmid pCRII using the TA-cloning kit (InVitrogen) and the nucleotidesequence of the insert was determined.

The nucleotide sequence of the resulting 300 bp RT-PCR product containedan open reading frame (ORF) with a deduced amino acid sequenceconfirming the sequences of the peptides from which the primers werederived as well as that of another of the sequenced peptides. The insertof this plasmid was radioactively labelled and used as a probe forscreening a total of 8×10⁵ clones in a commercial human liver library(Stratagene). Sixteen clones hybridized and the 4 longest (phl-1, 2, 3and 4) were completely sequenced. Sequence analysis revealed that allfour clones represent reverse transcripts of the same novel human mRNAspecies. The longest clone, phl-4, comprises 2475 bp starting with a 5′untranslated region of 36 bp followed by an ORF of 2061 bp and a 3′untranslated region of 378 bp ending with a poly-A tail. The nucleotidesequence (SEQ ID NO:3) of phl-4 is shown in FIG. 6 together with thetranslated amino acid sequence (SEQ ID NO:2). The sequences aredeposited at the EMBL nucleotide sequence data base (accession no.Y09926). While the sequence of phl-1 and -2 were in total agreement withphl-4, the nucleotide sequence of phl-3 differs from phl-4 at twopositions, a transversion at nucleotide position 1147 (G to T) and atransition at position 1515 (C to T). The first change leads to thereplacement of Asp 356 with Tyr. Because all clones were isolated from aliver library transcribed from RNA isolated from a single donor, theobserved difference may represent a polymorphism in the MASP-2 gene, oris due to an error created during construction of the library.

The amino acid sequences of the NH₂-terminus as well as all sequencedpeptides were identified in the sequence deduced from clone ph1-4. TheORF encodes a polypeptide chain of 686 amino acids (SEQ ID NO:2)including a signal peptide of residues. Omitting the signal peptide, thecalculated M_(r) is 74,153, in agreement with the 76 kDa observed onSDS-PAGE (FIG. 1), the isoelectric point is 5.43 and the molarextinction coefficient is 113,640 (i.e. OD_(280 nm)=1.54 at 1 mg/ml). Incontrast to MASP-1 (SEQ ID NO:6) the sequence contains no sites forN-linked glycosylation. The three amino acid residues which areessential for the active centre in serine proteases (His 468, Asp 517,and Ser 618) are present.

Example 5 Comparison of MASP-2 to MASP-1, C1r and C1s

The amino acid sequence deduced from the cDNA sequences is homologous tothose of MASP-1 (SEQ ID NO:6), C1r (SEQ ID NO:7) and C1s (SEQ ID NO:8)(FIG. 2). Notably, the domain organization is common to these fourproteins, featuring one C1r/C1s-like domain, one epidermal growthfactor-like (EGF-like) domain, followed by a second C1r/C1s-like domain,two complement control protein (CCP) domains, and a serine proteasedomain. The key residues involved in the calcium-binding motif in theepidermal growth factor-like domains are present in the obtainedsequence (SEQ ID NO:2), as well as in MASP-1 (SEQ ID NO:6), C1r (SEQ IDNO:7) and C1s (SEQ ID NO:8). In addition, the substrate specificityrelated residue, 6 residues before the active site serine, is asparticacid in all four proteins. MASP-1, C1r, and C1s are all activated bycleavage of the peptide bond between the residues Arg and Ile locatedbetween the second CCP domain and the serine protease domain. Theresulting polypeptide chains (the largest referred to as the “heavychain” and the smallest as “light chain”) are held together by adisulphide bond. By analogy, our results indicate that the 52 kDapolypeptide, recognized by antibody against the N-terminal of MASP-2after SDS-PAGE under reducing conditions, is the heavy chain of MASP-2,whereas the 31 kDa polypeptide, recognized by antibody against theC-terminal of MASP-2, is the light chain. As seen in FIG. 2, Arg and Ileare present in MASP-2 (SEQ ID NO:2) at the expected positions betweenthe second CCP domain and the protease domain.

Identities and similarities between the four proteins were studied basedon the alignment in FIG. 2. A bias of 6 was added to each term of themutation data matrix (250PAMS) and a break penalty of 6 was used.Identical residues in all four species are indicated by asterisks. Thebeginning of the C1r/C1s-like domains, the EGF-like domain and the CCPdomains are indicated above the sequences. The aligned cysteines areshaded. The potential cleavage site between Arg and Ile residues, whichgenerates heavy and light chains, is identical to the site where theserine protease domain starts. The three amino acid residues, which areessential for the active centre in serine proteases (His 468, Asp 517and Ser 618), are indicated (♦). The cysteines in the histidine-loop ofMASP-1 (SEQ ID NO:6) are marked (∇). The sequences obtained by aminoacid sequencing of peptides are underlined. The identities between theproteins (FIG. 2) are all in the range of 39% to 45% and gives no clueto functional relatedness. The similarity, i.e. taking into accountresidues of similar nature as well as identical residues, between theproteins (FIG. 3 b) are between 39 and 52% with the least similaritybeing between MASP-1 (SEQ ID NO:6) and C1s (SEQ ID NO:8) (39%) and thehighest similarity between MASP-1 (SEQ ID NO:6) and C1r (SEQ ID NO:7)(52%) and between MASP-1 (SEQ ID NO:6) and MASP-2 (SEQ ID NO:2) (52%).MASP-2 (SEQ ID NO:2) shows similarity with C1r (46%) (SEQ ID NO:7) andC1s (47%) (SEQ ID NO:8). Whereas the relative identities gives no clueas to functional relatedness the similarity score between C1s and MASP-2is significantly higher than between C1s and MASP-1 while MASP-1 is moresimilar to C1r than to C1s, suggesting that MASP-2, like C1s, could be aC2 and C4 cleaving enzyme. Several features of the sequences suggestthat MASP-2, C1r and C1s have evolved by gene duplication and divergencefrom a MASP-1 ancestor. Only the MASP-1 (SEQ ID NO:6) sequence containsthe histidine loop, characteristic of trypsin-like serine proteases²⁷.The active site serine is encoded by a TCN codon (N is A, T, G or C) inMASP-1 as in most serine proteases, whereas in MASP-2 (SEQ ID NO:2), C1r(SEQ ID NO:7) and C1s (SEQ ID NO:8) it is encoded by an AGY codon (whereY is T or C). In most serine proteases, including MASP-1 (SEQ ID NO:6),a proline residue is found at the third position downstream from theactive site serine, whereas a different amino acid is found in MASP-2(SEQ ID NO:2), C1s (SEQ ID NO:8) and C1r (SEQ ID NO:7) (alanine inMASP-2 and C1s, valine in C1r). Based on these analogies one may predictthat the catalytic domain of MASP-2 is encoded by a single exon as inC1r and C1s, whereas most other serine proteases, including MASP-1²⁸(SEQ ID NO:6), have split exons.

Example 6 MBL/MASP Complexes

The lectin preparation described above was incubated in microtiter wellscoated with monoclonal anti-MBL antibody, or, as a negative control,wells coated with non-specific monoclonal immunoglobulin of the samesubclass. The proteins captured by the antibody were eluted and analyzedby SDS-PAGE/Western blotting. The results (FIG. 3 a 1 to 3 a 3) showthat the anti-MBL antibody, in addition to binding MBL, captures bothMASP-1 and MASP-2. Microtiter wells were coated with monoclonal anti-MBLor control monoclonal murine IgG1, incubated with either one of twodifferent lectin preparations (a and b), and the bound proteins wereeluted and analyzed by SDS-PAGE under reducing conditions and Westernblotting. Blot a (FIG. 3 a 1) was developed with anti-MBL antibody, blotb (FIG. 3 a 2) with anti-C′ MASP-1 antibody and blot c (FIG. 3 a 3) withanti-N′ MASP-2 antibody. Lane I represents unfractionated lectinpreparation a. Lanes 3 and 4 represent eluates from wells coated withanti-MBL antibody and incubated with lectin preparation b and a,respectively, while lanes 2 and 5 represent eluates from wells coatedwith normal IgG and incubated with lectin preparation b and a,respectively.

Fractions from gel permeation chromatography (GPC) of the lectinpreparation on Superose 6B CL were analyzed from MBL, MASP-1 and MASP-2(FIG. 3 a 1 to 3 a 3). The lectin preparation was subjected to GPC on aSuperose 6 column in buffer containing calcium. MBL was eluted in a mainpeak at a volume (V_(e)) corresponding to an M_(r) of 750 kDa, and asmaller peak at a position corresponding to 350 kDa. FIG. 3 b 1 showsthe results of analysis of the fractions by Western blotting usingmonoclonal anti-MBL antibody. The band at about 60 kDa is seen in allMBL preparations and is recognized by all the anti-MBL antibodies tested(monoclonal as well as polyclonal) and thus probably represents anon-reducible dimer of the 32 kDa polypeptide chain. FIG. 3 b 2 showsthe same analysis using anti-N′ MASP-2 antibody (developing the upperband of 52 kDa) followed by anti-C′ MASP-1 antibody (developing thelower band of 31 kDa). For purely technical reasons the 20 kDa truncatedMASP-2 is not seen in this picture where the blot was partially strippedbetween the incubations with anti-MASP-2 and anti-MASP-1. The arrows onthe chromatogram FIG. 3 b 3 indicate the void volume (1) and the elutionpositions for the following marker proteins IgM (2), C1q (3),thyroglobulin (4), IgG (5) and serum albumin (6).

MASP-1 and MASP-2 coelute largely with the high molecular weight MBL.Chromatography of the MBL preparation at pH 5 revealed that no MASP-1 orMASP-2 was associated with MBL. See, Tan et al. (1996, Biochem J.319:329-332).

Example 7 Complement Activation

The classical complement activation pathway, as well as theMBL-initiated pathway involves the generation of a C3 convertingcomplex, C4b2b, through enzymatic activation of C4 and C2. In the C1complex (C1qr₂ s₂) this specific protease activity is exhibited by C1safter activation of the enzyme by C1r. Upon activation of C4, a reactivethiol ester is exposed and C4b covalently binds to nearby amino orhydroxyl groups.

The C4 activating potentials of MASP-1 and MASP-2, and C1r and C1s, werecompared. This was accomplished by separating a C1 preparation and anMBL/MASP preparation by SDS-PAGE followed by Western blotting. The blotwas examined for C4 converting activity by incubation with human serumat 37° C., followed by detection of deposited C4b using anti-C4antibodies.

C1 was purified by incubating IgG-coupled Sepharose beads with humanserum at 4° C. The beads were washed and incubated at 37° C. for 30minutes for activation of C1r and C1s. The beads were suspended innon-reducing sample buffer and, without boiling, subjected to SDS-PAGE,followed by blotting in the absence of SDS. A similar blot was made ofan MBL preparation produced in the absence of enzyme inhibitors (TheState Serum Institute, Copenhagen). Strips of the blots were incubatedfor 30 minutes at 37° C. with 1.1% (v/v) human MBL-deficient serum,depleted of C1q by fractionation on Biorex 70. The blots were developedwith biotinylated F(ab′)₂ anti-C4 antibody followed byperoxidase-labelled streptavidin and luminescence reagent. Parallelblots were treated with a serine protease inhibitor(aminoethylbenzenesulfonyl fluoride), which was also present duringincubation with serum. Other strips were directly developed withantibodies.

The results in FIG. 4 show that C4 was deposited at a positioncorresponding to the MASP-2 band, whereas no C4 deposition was observedat positions corresponding to MASP-1. MASP-1 was present in theactivated state as shown by SDS-PAGE under reducing conditions where itappears as two bands at about 30 kDa and 70 kDa, respectively (notshown). The observed C4 activation and deposition was inhibited byserine protease inhibitors (FIG. 4). It was also observed that no C4activating activity could be detected when MBL/MASP was prepared in thepresence of enzyme inhibitors added throughout the purificationprocedure. A preparation of C1 was analyzed similarly and C4 deposition,which could be inhibited by enzyme inhibitors, was observed at aposition corresponding to C1r and C1s, which are not separated by thetechnique employed.

FIG. 4 is a representation of Western blots demonstrating the activationof C4 by C1s and MASP-2. Panel A shows a Western blot of C1 separatedunder non-reducing conditions, and without heating the sample beforeelectrophoresis. Lane 1 was developed with anti-C1s antibody. Lane 2 wasincubated with human serum followed by anti-C4 antibody. Lane 3 was aslane 2 except for the presence of serine protease inhibitors during theincubation with serum. Panel B shows a Western blot of an MBLpreparation separated as in A. Lane 1 was developed with anti-N′ MASP-1,lane 2 with anti-N′ MASP-2. Lane 3 was incubated with human serum at 37°C. followed by anti-C4. In lane 4 the blot was preincubated with serineprotease inhibitors and the incubation with serum was also in thepresence of inhibitors. MASP-1 shows a higher M_(r) than MASP-2 due toglycosylation¹⁷ and a polypeptide chain 9 amino acids longer.

Our results emphasize the similarity between complement activationthrough the MBLectin pathway of the innate immune system and theclassical pathway of complement activation (FIG. 5). Activation via theclassical pathway is associated with the specific immune response foundonly in vertebrates, while the MBLectin pathway and the alternativepathway rely on innate recognition of foreign organisms and are thuslikely to predate the evolution of the specific immune system. Allpathways converge on the activation of the central component C3 intoC3b, which binds covalently to the microbial surface and mediates theeffector functions of complement.

In both the classical and MBLectin pathways, the initiating molecularcomplexes are composed of an oligomeric ligand-binding component (MBL orC1q, respectively) which, on reacting with ligands, activates the twoassociated serine proteases (MASP-1 and MASP-2 or C1r and C1s,respectively).

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1. An isolated nucleic acid molecule capable of remaining hybridized toa nucleic acid sequence comprising SEQ ID NO:3, or to the antisensecomplement of a nucleic acid sequence comprising SEQ ID NO:3, understringent wash conditions.
 2. An isolated nucleic acid molecule of claim1, wherein said nucleic acid sequence encodes an amino acid sequence ofSEQ ID NO: 1 or portions thereof, or SEQ ID NO: 2 or portions thereof.3. The isolated nucleic acid molecule of claim 1 comprising the nucleicacid sequence of SEQ ID NO:3, or portions thereof.
 4. An isolatednucleic acid molecule that encodes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:
 2. 5. The isolated nucleic acid molecule of claim 4 thatencodes the amino acid sequence of SEQ ID NO:
 2. 6. An expression vectorcomprising the nucleic acid molecule of claim
 1. 7. The expressionvector of claim 6 operably associated with a regulatory element thatdirects the expression of the encoded amino acid molecule.
 8. A hostcell comprising the vector of claim
 7. 9. A method of producingrecombinant MASP-2 polypeptide comprising introducing the expressionvector of claim 6 into a host cell to create a recombinant host cell,growing the recombinant host cell under conditions where MASP-2polypeptide is expressed from said expression vector and recovering saidrecombinant MASP-2 polypeptide.
 10. An isolated recombinant polypeptidecomprising an amino acid sequence at least 95% identical to SEQ ID NO: 1or SEQ ID NO:
 2. 11. The isolated polypeptide of claim 10 comprising SEQID NO:
 1. 12. The isolated polypeptide of claim 10 comprising position16 to position 686 of SEQ ID NO:2.
 13. A substantially pure recombinantpolypeptide, which polypeptide comprises an amino acid sequence at least95% identical to the sequence defined by position 16 to position 686 ofSEQ ID NO:2; wherein said recombinant polypeptide has the followingactivities: i) cleavage of C4 and activation of the complement in an invitro assay for MBLectin complement pathway function; and ii)mannan-binding lectin (MBL) associating activity.
 14. A pharmaceuticalcomposition comprising the polypeptide of claim
 11. 15. A pharmaceuticalcomposition comprising the polypeptide of claim 12 or claim
 13. 16. Anisolated antibody or fragment thereof that specifically binds to apolypeptide comprising an amino acid sequence identical to SEQ ID NO: 1or SEQ ID NO:
 2. 17. An antibody that specifically binds to a humanmannan-binding lectin associated serine protease-2 (MASP-2).
 18. Theantibody of claim 16 or 17, wherein the antibody specifically binds to apolypeptide comprising position 16 to position 686 of SEQ ID NO:2. 19.The antibody of claim 18, wherein the antibody is a monoclonal antibody.20. The antibody of claim 18, wherein the antibody is a chimericantibody.
 21. The antibody of claim 18, wherein the antibody is asingle-chain antibody.
 22. A pharmaceutical composition comprising theantibody of claim 16 or 17.